The mesencephalic astrocyte-derived neurotrophic factor (MANF) has been described as a survival factor for dopaminergic neurons in vitro (Petrova et al., 2003; WO 01/19851; and WO 02/074956), but its expression in mammalian tissues is poorly known. We recently characterized a homologous protein, the conserved dopamine neurotrophic factor (CDNF), which protects and rescues dopaminergic neurons in vivo (Lindholm et al., 2007; WO 2007/068784; and WO 2007/068803). MANF and CDNF proteins form a novel evolutionary conserved family of neurotrophic factors.
Neurotrophic factors are secreted proteins that promote neuronal survival and enhance formation and maintenance of neuronal connections in the vertebrate nervous system (Huang and Reichardt, 2001; Airaksinen and Saarma, 2002). The role of neurotrophic factors is well characterized mainly in the peripheral nervous system (PNS). In recent years, substantial knowledge has accumulated also about the involvement of growth factors in regulating the development and maintenance of neuronal populations in the central nervous system (CNS).
The characteristic motor symptoms of Parkinson's disease (PD) result from gradual degeneration of midbrain dopaminergic neurons. Several growth factors can act as survival factors for dopaminergic neurons (Krieglstein, 2004; Bespalov and Saarma, 2007), but their roles in the development and maintenance of these neurons in vivo are still unknown. Glial cell line-derived neurotrophic factor (GDNF; Lin et al., 1993) protects and even repairs the dopaminergic system in rodent and primate models of PD (Hoffer et al., 1994; Sauer et al., 1995; Kearns and Gash, 1995; Tomac et al., 1995; Gash et al., 1996; Grondin et al., 2002). However, results from clinical trials with GDNF on Parkinsonian patients have been controversial, since either clinical improvement (Gill et al., 2003; Patel et al., 2005) or low clinical efficacy (Lang et al., 2006) has been reported. Therefore, identification and characterization of new factors which could be neuroprotective and support functional neurorestoration is highly warranted.
Ischemic and epileptic insults, which are associated with neuronal death, trigger changes in the expression of several neurotrophic factors in the brain such as the GDNF family (Kokaia et al., 1999; Arvidsson et al., 2001) and the neurotrophins, e.g., BDNF (Ernfors et al., 1991; Lindvall et al., 1992). Changes in neurotrophic factor levels following brain insults may regulate cell survival, synaptic plasticity, epileptogenesis, and neurogenesis from endogenous neural stem cells (Binder et al., 2001; Gustafsson et al., 2003; Jin et al., 2003; Kuipers and Bramham, 2006; Schäbitz et al., 2007). Therefore, modulation of neurotrophic factor levels could become of therapeutic value in ischemic and epileptic conditions.
A novel neurotrophic factor mesencephalic-astrocyte-derived neurotrophic factor (MANF; also known as arginine-rich, mutated in early stage of tumors; ARMET) was described few years ago as a survival-promoting factor for embryonic midbrain dopaminergic neurons in vitro (Petrova et al., 2003). The observed survival promoting effect of MANF was specific for dopaminergic neurons, and no effects on serotonergic or GABAergic neurons were detected (Petrova et al., 2003). Our group has recently characterized a vertebrate-specific paralog of MANF, named the conserved dopamine neurotophic factor (CDNF). CDNF (ARMET-like1) protects and rescues midbrain dopaminergic neurons in vivo in a rat 6-OHDA model of Parkinson's disease (Lindholm et al., 2007). MANF and CDNF proteins form a new family of evolutionarily conserved factors (Lindholm et al., 2007). Importantly, the effects of MANF and CDNF on dopaminergic neurons suggest that these factors could be used in treatment of neurodegerative disorders (Petrova et al., 2003, Lindholm et al., 2007). However, in vivo data of MANF are still lacking. Moreover, it should be noted that based on EST and genomic sequences of MANF we concluded that in contrast to the sequence reported by Petrova et al. (2003), WO 01/19851 and WO 02/074956, the amino acid 176 of human MANF is arginine (R) instead of proline (P). In this light, it seems that the some of the previous works related to MANF have been made with a MANF polypeptide having an amino acid substitution at position 176, and results of these experiments are thus not relevant to the present invention (see also Example 9).
In this invention, we characterized the distribution of Manf mRNA and protein in developing and adult brain and in non-neuronal tissues. We also discovered the effect of MANF in pathological conditions, such as Parkinson's disease, status epilepticus and global forebrain ischemia (i.e. stroke).